Process for production of fibrinogen and fibrinogen produced thereby

ABSTRACT

A process for purifying fibrinogen from a fibrinogen containing source by precipitation of fibrinogen by a precipitating agent from a fibrinogen containing solution in the presence of one or more chelating agent(s) and removal of the supernatant from the fibrinogen paste, characterised in that fibrinogen is extracted from the paste forming a liquid fraction containing fibrinogen, and an undissolved residue, which is separated from the liquid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.14/382,712, filed Sep. 3, 2014, which is a U.S. National Stage ofInternational Application No. PCT/EP2013054983, filed Mar. 12, 2013,which claims priority to U.S. Provisional Application No. 61/610,030,filed Mar. 13, 2012, and European Application No. 12159276.0 filed Mar.13, 2012, the entire contents of each of which are hereby incorporatedby reference.

DESCRIPTION

The present invention relates to a process for purifying fibrinogen froma fibrinogen containing source, a fibrinogen product obtainableaccording to the process of the invention as well as an anion exchangeresin selected from the group consisting of a support materialcomprising a hydroxylated polymer backbone with grafted tertiary orquaternary amino groups for purification or manufacturing of afibrinogen product.

BACKGROUND OF THE INVENTION

Fibrinogen, also known as clotting factor I, plays a key role inhaemostasis and wound healing. It is a glycoprotein synthesized in theliver with an apparent molecular weight of 340,000 Da, is composed oftwo dimers, each of them built of three pairs of non-identicalpolypeptide chains called Aα, Bβ and γ linked by disulfide bridges. Itcirculates in the blood stream at a concentration of approximately1.5-4.0 mg/ml. Upon injury of blood vessels, blood platelets areactivated and a plug is formed. Fibrinogen is involved in primaryhaemostasis by aiding cross-linking of activated platelets.

In parallel activation of the clotting cascade is initiated. As theendpoint, fibrinogen is converted into fibrin by proteolytic release offibrinopeptide A and—at a slower rate—fibrinopeptide B by thrombin. Thesoluble fibrin monomers are assembled to double stranded twistedfibrils. Subsequently these fibrils are arranged in a lateral manner,resulting in thicker fibers. These fibers are then cross-linked byFXIIIa to a fibrin network, which stabilizes the platelet plug byinteractions of the fibrin with activated platelets, resulting in astable clot.

Disorders and Deficiencies

Congenital afibrinogenaemia is a rare bleeding disorder, where patientsare suffering from inadequate clotting of the blood due to lacking ormalfunction of fibrinogen. This medical condition might lead tospontaneous bleeding episodes or excessive bleeding after minor traumataor during interventional procedures.

Acquired deficiencies in fibrinogen are much more common than congenitalafibrinogenaemia and may be induced by haemodilution or other eventssuch as blood losses during surgery, traumata, disseminatedintravascular coagulation (DIC) or sepsis.

Fibrinogen deficiencies can be corrected to normal fibrinogen levels inplasma of about 1.5-4 g/l by replacement therapy with intravenousinfusion of fresh frozen plasma or cryoprecipitate. However, thesetreatments are afflicted with the risk of introduction of pathogens,e.g. viruses or prions, into a patient and are thereby generatingadditional disorders. It is thus advisable to intravenously apply virusinactivated fibrinogen compositions to restore fibrinogen atphysiological levels in a save way.

While there exists fibrinogen in preparations called fibrin glue,fibrinogen adhesive, tissue glue and similar, these preparations areintended for topical use as powders, pastes, foams or in combinationwith fabrics as plaster on wounds, they are not usable for intravenousapplication as their consistency and composition would immediatelyinitiate thrombotic events when being injected. These preparationsadditionally contain thrombin, calcium salts and relatively high amountsof coagulation factor XIII. Examples for such preparations areUS-A1-2008/003272, WO-A-95/22316 or US-A1-2008/181878.

Processes for fibrinogen production are known from EP-A1-1 240 200 whichrelates to a method of purifying fibrinogen from a fibrinogen containingsolution, comprising, application of a fibrinogen containing solution toan ion exchange matrix, under conditions such that fibrinogen binds tothe matrix, washing the ion exchange matrix with a buffer solutioncomprising at least one ω-amino acid, eluting the fibrinogen from thematrix with a buffer consisting of 10 mM Tris, 10 mM citrate, 45 mMsucrose and NaCl at a concentration of 200 mM to 1.0 M, and optionallyrecovering the fibrinogen from the eluate.

EP-A1-0 771 324 refers to a process for production of a virus freefibrinogen concentrate which is obtained by subjecting a solubilisedplasma fraction containing fibrinogen to a chemical viral inactivationtreatment, i.e. a S/D or solvent/detergent treatment, subjecting theresulting viral-inactivated fraction to precipitation in a solutioncontaining an amino acid at an acidic pH to obtain a supernatant,filtering the supernatant to obtain a purified fibrinogen concentrate,and recovering the purified fibrinogen concentrate. The recoveredfibrinogen concentrate is subjected to ultra violet radiation for asecond virus inactivation. The product is stabilized and lyophilizedprior to a third virus inactivation step.

EP-A1-1 519 944 teaches the use of an immobilized metal ion affinitychromatography matrix under conditions that the fibrinogen andplasminogen bind to the matrix, and selectively eluting the fibrinogenand 93% of plasminogen separately from the matrix.

EP-A1-0 555 135 discloses a method for production of an intravenouslyapplicable fibrinogen by purification of a fibrinogen solution by meansof an anion exchange gel based on cross-linked agarose comprisingquaternary amine groups. The fibrinogen produced is said to be free offactor VIIIc.

EP-A1-1 457 497 refers to a process for removing viruses in fibrinogensolutions characterized by stabilization and freezing of the solutionand subsequent thawing thereof. Separation of undissolved materialsoccurs prior to dilution of the protein and is followed bynanofiltration of the resultant solution using filters of a pore sizesmaller than 35 nm.

US-A1-2006/0009376 also discloses a method to manufacture fibrinogen,following repeated dissolution and precipitation of fibrinogen to removefactor XIII.

WO-A2-2009/155626 refers to a process for purification of fibrinogen bysolubilisation of cryoprecipitate or Cohn-Fraction I in an EDTA-solutionat a temperature range of 3° C. to 5° C., followed by fractionatedprecipitation in a temperature range of 2° C. to 4° C. and dissolutionof the last precipitate in an EDTA-solution. Virus inactivation with S/Dreagents and nanofiltration for improvement of pathogen safety occur inthe presence of EDTA. Inhibition of residual amounts of proteases in theproduced concentrate is accomplished by addition of AT-III,heparin-cofactor-II and C1-esterase-inhibitor.

U.S. Pat. No. 7,919,592 B2 describes a method for removing viruses fromfibrinogen solutions by addition of chaotropic substances, chosen fromarginine, guanidine, citrulline and urea, there salts or derivatives andsubsequent filtration through nanofilters of various pore size.

Goheen, S. C. et al. report in Journal of Chromatography A. 816 (1998)89-96, about HPLC ion-exchange chromatography of the plasma proteinsalbumin, fibrinogen, and immunoglobulin (G) on nonporous columnmaterials containing either quaternary amine or sulfopropyl functionalgroups.

SUMMARY OF THE INVENTION

One object of the invention is providing of a fibrinogen concentratemanufactured with dedicated pathogen eliminating and/or inactivationsteps in the production process in order to overcome adverse reactionsor development of pathogen related illnesses. Said pathogens areselected from the groups of bacteria, viruses and prions, such as prionprotein scrapie (PrP^(sc)).

Nanofiltration is a method in principle known to remove viruses fromproteins passing through the nanofilter, but separation of viruses fromfibrinogen by nanofiltration is challenging as fibrinogen is a quitelarge and sticky protein frequently resulting in plugging of filterpores and eventual loss of product. One approach to overcome thisproblem is to add chaotropic substances to improve filterability as itis taught by U.S. Pat. No. 7,919,592B-2, although nanofiltration of adiluted fibrinogen solution produced according to the process ofInternational Application WO-A1-2012/038410 revealed comparablefilterability without addition of chaotropic substances. Chaotropicsubstances according to the invention are those as defined in U.S. Pat.No. 7,919,592 B2 incorporated by reference and designating arginine,guanidine, citrulline and urea, their salts or derivatives.

A further object of this application is providing of a process tomanufacture a concentrate on an industrial level, i.e. several hundredsto thousands of liters starting material, such as blood or blood plasma,although a small scale production, i.e. some 1/10 liter to some liters,is also possible.

These and further objects are accomplished by a process of claims 1 to18, a product as claimed in claims 19 to 24 obtainable by the process ofthe invention and the use of claim 25.

FIG. 1 depicts a SDS-Page at non-reducing conditions while FIG. 2depicts a SDS-Page at reducing conditions of the same samples.

According to the present invention it has surprisingly been observedthat precipitation of an intermediate fibrinogen paste in the presenceof one or more chelating substances followed by extraction of fibrinogenfrom the intermediate provided a fibrinogen solution of betterfilterability than those produced by the process of WO-A1-2012/038410.

It was further surprisingly observed, that a one-time addition of arather small amount of at least one chelating agent prior toprecipitation, resulting in a total chelating agent concentration ofabout 3-10 mM, is sufficient for the present invention to provide bothexcellent yield and filterability, even with nanofilters providingretention of particles of <35 nm. Residual amounts of chelating agentcan be removed downstream providing a final product, i.e. a fibrinogenconcentrate which is free of chelating agent within the detection limit,which is lower than 0.08 μg/ml for EDTA. A product free of chelatingagents is preferable as, for instance, EDTA is a known anti-coagulant.Thus the presence of EDTA in substantial amounts is counterproductive tothe effects of a fibrinogen product.

Consequently, all buffers used downstream of the precipitation, e.g.equilibration-, washing- and elution-buffers used for chromatography orthe buffer used during concentration by ultra-/diafiltration, should befree of Ca²⁺-chelating agents, which are defined below. Residual amountsof eventually present chelating agents are removable from a fibrinogencontaining solution by ultra-/diafiltration. This removal isadvantageously performed by ultra-/diafiltration at the end of theprocess in particular during concentrating or formulating, if it shouldbe necessary. Systemic application of such a fibrinogen product free ofchelating agents via intravenous route allows treatment of congenitalafibrinogenaemia and acquired fibrinogen deficiencies. Application ofthis standardized fibrinogen concentrate allows fast treatment inemergency situations without time-consuming thawing of fresh frozenplasma and lowered volume load and reliable coagulation properties dueto essentially constant composition. Increased concentrations ofcoagulation factor XIII, compared to products disclosed inWO-A1-2012/038410 also support topical administration on wounds or theuse as tissue glue.

It was further surprisingly observed, that an addition of proteaseinhibitors at any step of the process of the invention was unnecessarywhen the intermediate fibrinogen paste was precipitated in the presenceof one or more chelating substances. The use of protease inhibitors suchas C1-protease inhibitors, trypsin inhibitors, thrombin inhibitors,antithrombin-III (AT-III), heparin-cofactor-II, aprotinin, pepstatin,leupeptin and in particular epsilon-aminocaproic acid (ε-ACA) to avoiddegradation of fibrinogen is known from prior art literature. Thefibrinogen concentrate of the present invention displayed neither ameasurable proteolytic activity nor a measurable AT-III concentration.

In general, the process of the invention for purification ormanufacturing of fibrinogen from fibrinogen containing sources comprisesthe steps of:

-   -   forming a fibrinogen enriched precipitate by adding a chelating        agent prior to addition of at least one precipitating agent to        the fibrinogen containing source;    -   isolating the fibrinogen enriched precipitate e.g. by        centrifugation of said precipitate;    -   extraction of fibrinogen from the fibrinogen enriched        precipitate in an aqueous medium void of the chelating agent and        thereby forming a fibrinogen containing solution, optionally        followed by filtration and/or ultra/diafiltration;    -   subjecting the fibrinogen containing solution to a        chromatography on a stationary phase having strong anion        exchanger groups by contacting said solution with said phase        under conditions that fibrinogen binds to said phase;    -   followed by an elution of fibrinogen from the stationary phase        by means of an aqueous solution having a higher ionic strength        than the ionic strength of the foregoing step, yielding a        fibrinogen enriched fraction which is collected;    -   optionally followed by subsequent steps of dilution and/or        concentration of the fibrinogen enriched fraction;    -   and optionally filling of the fibrinogen enriched fraction into        suitable vials, while addition of protease inhibitors is        omitted.

In one embodiment of the manufacturing process of the invention thefibrinogen containing source is selected from the group consisting of,blood plasma, plasma fractions, such as fraction I, or cryoprecipitate,cell cultures producing fibrinogen and/or supernatants of said cellcultures. If cryoprecipitate is not used as starting material, afibrinogen containing intermediate as starting material is produced bywell known methods like disclosed by Cohn, Kistler-Nitschmann andmodifications thereof.

For obtaining a pharmaceutical usable product it is advantageous thatthe fibrinogen containing source is subjected to at least one virusinactivating process for example a solvent detergent process asdisclosed in EP-A1-0 131 740 incorporated by reference.

According to another embodiment of the invention the virus inactivationis performed prior to forming a fibrinogen enriched precipitate.However, it is also possible to perform a virus inactivation at adifferent stage.

According to yet another embodiment of the invention the removal ofvirus inactivating substances is performed by oil extraction and/orchromatography with strong anion exchangers. Another method for virusremoval is nanofiltration, due to the low content of polymers afterfibrinogen extraction nanofiltration can be performed also well withfilters<35 nm without addition of chaotropic substances.

A typical precipitating agent for use in the manufacturing process ofthe invention is selected from the group consisting of amino acids suchas glycine, polyethylene glycol or high salt concentrations, wherein thesalt contains monovalent metal ions, in particular selected from thegroup of alkali metals, or ammonium.

According to still another embodiment of the invention the extraction offibrinogen from the fibrinogen enriched precipitate is performed with abuffer having a pH of 7.5 to 8.5 for 10 to 120 minutes.

According to a further embodiment of the invention the stationary phasehas tertiary or quaternary amino groups.

The chromatography steps in the manufacturing process of the inventioncan in particular be performed in a column.

Typically the storage form of the filled fibrinogen enriched fraction isin liquid state, frozen state, preferably at <−15° C., most preferablybelow −30° C., or as lyophilisate.

The process of the invention comprises in detail the steps of

-   -   a) solubilisation of cryoprecipitate, solubilised at about        neutral pH,    -   b) subjecting the solution to adsorption with Al(OH)₃ and        removing the resulting gel,    -   c) virus inactivating the resulting solution of step b) by a        solvent/detergent (S/D) treatment, extraction of S/D reagents        with vegetable oil and contacting the water-phase with a TMAE        resin,    -   d) addition of at least one chelating agent to obtain a        chelating agent concentration for example of 3 mM to 100 mM in        the resulting water phase of step c),    -   e) precipitation of fibrinogen from the chelating agent        containing water phase from step d), by adding glycine until a        final concentration of about 1M glycine is reached, and        separating of the resulting fibrinogen paste,    -   f) extraction of fibrinogen from the fibrinogen paste by a 20 mM        TRIS buffer at a pH of about 8.0, filtration and,    -   g) loading the filtered solution of step f) onto an anion        exchange resin comprising trimethyl-amino groups grafted to a        hydroxylated methacrylic polymer backbone via linking groups and        washing off loosely bound substances with a wash buffer of about        12.0 mS/cm conductivity,    -   h) elution of fibrinogen with an elution buffer containing about        1.5 g/l sodium citrate, about 7.0 g/l sodium chloride and about        10.0 g/l glycine, in particular adjusted to a pH of about 7.0        and a conductivity of 13.1-15 mS/cm,    -   i) filtering over at least one nanofilter    -   j) concentrating, formulating, sterile filtering, filling into        suitable containers, and optionally lyophilisation.

Subject matter of the present invention is also a fibrinogen enrichedfraction obtainable according to the manufacturing process of theinvention.

The fibrinogen concentrate is filled into final containers after sterilefiltration and it may be stored in liquid, liquid frozen or lyophilisedform. Suitable containers are glass vials or bottles or plastic bagseventually comprising a membrane allowing lyophilisation while the bagis tightly sealed off for fluids.

Fibrinogen produced according to this process is characterised by verylow amounts of impurities which ascertain the nativity of the productand allows long term treatment of people in need. FXIII is preferable atthe concentration contained, because it supports stabilisation of theformed fibrin, while an overload of this transglutaminase is avoided.

The term “comprising”, “comprise” or “comprises” can also be replaced by“consisting”, “consist” or “consists” without altering the disclosure ofthe description.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts a SDS-Page at non-reducing conditions of productsproduced according to the process of the present invention (Lanes 6-11also indicated as “+”) and the product of patent applicationWO-A1-2012/038410 (Lanes 2-5 also indicated as “−”).

FIG. 2 depicts a SDS-Page at reducing conditions of products producedaccording to the process of the present invention (Lanes 6-11 alsoindicated as “+”) and the product of patent applicationWO-A1-2012/038410 (Lanes 2-5 also indicated as “−”).

DETAILED DESCRIPTION

Although in principle all fibrinogen containing sources can be usedaccording to the invention, cryoprecipitate is a preferred source and inthe following the cryoprecipitate serves as a typical source offibrinogen in the further description of the manufacturing process ofthe invention.

Typically cryoprecipitate is reconstituted or solubilised under suitablebuffer conditions in particular at about neutral pH (6.9-7.0 for examplein a solution buffer containing Na-citrate and NaCl), subjected toadsorption in particular with Al(OH)₃ and the resulting gel removed e.g.by centrifugation. The supernatant can then become virus inactivated forexample by solvent/detergent (S/D) treatment. This method is well knownto the skilled person and has been originally described in EP-A1-131740. S/D compounds such as Triton (O-[4-(1,1,3,3-Tetramethylbutyl)phenoxy]-polyethoxyethanole) and TnBP (Tri-n-butyl-phosphate) are inparticular removed by extraction with castor oil. For furtherpurification the water-phase can be subjected to a chromatographicprocess. Typically this can be performed by contacting the water-phasewith a strong anion-exchange gel, tri-methyl-amino-ethyl (TMAE) graftedon matrix material, such as Fractogel® EMD-TMAE. Good results areachievable if the chromatography is performed with buffers having apH-value of 6.9-7.1 and an osmolality of 570-610 mosmol/l. Under theseconditions fibrinogen is not bound to the stationary phase and hencefound in the flow-through or supernatant, the latter if abatch-chromatography process is performed.

Unbound fibrinogen solution, containing typically about 40 g/l (Clauss'turbidometric method) is adjusted to pH=7.0-8.0, in particular topH=7.3-7.5, with a buffer containing at least one chelating agent.Suitable chelating agents are Ca²⁺-chelating agents in particular1,2-bis(o-amino)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),diethylene-triamine-pentaacetic acid (DTPA), ethylenediamine-tetraaceticacid (EDTA), ethylene-glycol-tetraacetic acid (EGTA) andnitrilo-triacetic acid (NTA) at concentrations of 3 mM to 100 mM, inparticular of 5 mM to 50 mM, even more particular of 5 mM to 20 mM.Thereafter a suitable precipitating agent, for example glycine, is addedto end up at a concentration of 0.8-1.2 M, in particular 0.9-1.1M theresulting solution can be stirred for 60-120 min to precipitatefibrinogen. Precipitation can be performed in a temperature range of+4.1° C. to +40° C., as long as cryoprecipitation is omitted, inparticular in the range of +5° C. to 37° C., more particular from 5.1°C. to 30° C., even more particular at 10°-20° C. The fibrinogencontaining precipitate can then be separated by centrifugation and thisintermediate fibrinogen paste might be stored at ≤−60° C., preferably at−100° C. to −65° C., for at least 6 months, if the intermediatefibrinogen paste is not processed without delay, but a storage time of 1day up to 6 months is preferred. Already a single precipitation e.g.with glycine provides a fibrinogen paste sufficiently pure for furtherprocessing.

Fibrinogen is then extracted from thus prepared intermediate by a 10-30mM tris(hydroxymethyl)aminomethane buffer (Tris buffer) free ofchelating agent at a pH value from 7.5 to 8.5, in particular a 15-25 mMTris buffer with pH=7.5-8.5. Extraction takes place for 10-120 minutes,in particular for 15-90 minutes, even more particular for 20-60 minutesduring stirring. The suspension obtained can then be filtered off andsubjected to ultra/diafiltration for example against 5 times of thesuspension volume of the same or a different buffer.

The resulting fibrinogen containing solution is then loaded onto ananion-exchange gel preferably selected from a group of tertiary orquaternary amino groups as ligands grafted to a matrix. Said functionalgroups are selected from well known diethyl-amino-ethyl (DEAE) or, inthe case of a strong anion exchange gel, from groups such astri-methyl-amino, tri-methyl-amino-ethyl (TMAE) and other groups whereasthe carrier material may be composed of cellulose, agarose, silica,polymeric or ceramic material. Good results, in particular in thereduction of fibronectin and vitronectin, can be achieved withtrimethyl-amino groups grafted to a hydroxylated methacrylic polymer viaa linking group such as GigaCap Q-650M®. This is very surprising as thechemically similar Marco-Prep High Q®, a methacrylic copolymer composedof diethylene-glycol-dimethacrylate/glycidyl-methacrylate also withtrimethyl-amino ligands but misses the hydroxyl functionality in itspolymeric backbone, is less efficient in the reduction of said twoproteins. The effective reduction of the sticky fibronectin is veryadvantageous for optional filtrations, such as ultra/diafiltration ornanofiltration, as the lifetime of filters is increased due to reducedclogging. If the process is intended to include nanofiltration, it ispreferred to perform the process with a diluted solution, in particularwith a cascade of nanofilters. The chromatographic gel or resin is inparticular preequilibrated with the same buffer as used for resuspendingthe intermediate fibrinogen paste before applying the fibrinogensolution. Loosely bound substances were washed out with equilibrationbuffer followed by washing buffer (1.5 g/l sodium citrate, 6.0 g/lsodium chloride, adjusted to pH=6.8-7.2, preferably 6.9-7.1, andpossessing the conductivity of 11.0-13.0 mS/cm at room temperature of20-25° C.).

Fibrinogen can then be eluted from the chromatographic column with anelution buffer containing 1.5 g/l sodium citrate, and 10.0 g/l glycinein particular adjusted to the same pH range as the washing buffer e.g.by HCl and/or NaOH and adjusted with about 7.0 g/l NaCl to theconductivity of 13.1-15 mS/cm at room temperature of 20° C.-25° C.Approximately 74% of the fibrinogen applied onto the column is recoveredin the eluate, whilst fibronectin is almost completely removed from thefibrinogen containing eluate. Advantageously a filtration in particulara nanofiltration is performed.

This filtered fibrinogen solution can further be concentrated byultra/diafiltration to about 20-26 g/l and sterile filtered withmembranes of ≤0.2 μm nominal pore size. Persons skilled in the art knowthat other concentrations, such as 1-19.9 g/l or 26.01-30 g/l or evenhigher are also achievable. The fibrinogen concentrate of the presentinvention may also be formulated with additives like stabilisers knownby the skilled person such as carbohydrates, e.g. sucrose, trehalose,amino acids, e.g. glycine, histidine, alanine, arginine and detergents,e.g. polyoxyethylene-(20)-sorbitan-monooleate (TWEEN 80®). This sterilefiltered bulk is stored at −60° C. or lower, in particular at −65° C. to−80° C., before being sterile filtered for a second time and filled intofinal containers and optionally freeze dried or directly filled intofinal containers and optionally freeze dried without a second sterilefiltration.

It is not necessary to add further buffers, stabilisers, proteaseinhibitors, like AT-III, heparin-cofactor-II and C1-esterase-inhibitor,or other compounds, like coagulation factor XIII (F XIII). Coagulationfactor XIII is present in the concentrate with activities of ≥0.05 IUper mg fibrinogen (Clauss method), in particular with activities of0.05-0.30 IU/mg. The fibrinogen concentrate of the present invention isfurther characterised by a low content of compounds of higher molecularweight than fibrinogen (HMW), determined as % of total area by sizeexclusion chromatography at 280 nm. The fibrinogen concentrate of thepresent invention contains less than 11% HMW, in particular 2-10% whenthe concentration of the chelating agent was at least 3 mmol/l. The useof chelating agents at concentrations of at least 5 mmol/l reduced theHMW-content to 2-6%. Some albumin may also be present in a concentrationof about 16 ng per mg fibrinogen. Antithrombin-III (AT-III) andproteolytic activity were not detectible, i.e. an AT-III concentrationof less than 0.2 IU/ml and a proteolytic activity of less than 2 U perliter (<2 U/l), which equates to less than 0.01 IU AT-III per mgfibrinogen and less than 0.1 mU proteolytic activity per mg fibrinogen,when being measured in a solution of the final product containingfibrinogen in a concentration of 20 mg/ml. The invention is furtherexplained by the following non-limiting examples.

EXAMPLE I

Cryoprecipitate, produced from plasma by established methods, wasreconstituted or solubilised at about neutral pH, subjected toadsorption with Al(OH)₃ and the resulting gel removed by centrifugation.The supernatant was then virus inactivated by solvent/detergent (S/D)treatment. S/D compounds, according to EP-A1-0 131 740 were extractedwith vegetable oil and the water-phase was contacted with Fractogel®EMD-TMAE. Chromatographic conditions (pH-value of 6.9-7.1 and anosmolality of 570-610 mosmol/l) were employed under which the fibrinogendid not bind to the gel and hence was found in the flow-through orsupernatant.

The solution of unbound fibrinogen was admixed with EDTA until the EDTAconcentration reached 10 mM and the EDTA containing fibrinogen solutionwas stirred at about 15° C. for about 60 minutes after addition ofglycine (1 mol/l final concentration and pH=7.4) to precipitatefibrinogen. The fibrinogen containing precipitate was then separated bycentrifugation, yielding an intermediate fibrinogen paste.

The fibrinogen was extracted by stirring for about 30 minutes from thusprepared intermediate by a 20 mM Tris buffer (pH=about 8.0) lacking achelating agent and the suspension obtained was then filtered andsubjected to ultra/diafiltration.

The resulting fibrinogen containing solution was then loaded ontoGigaCap Q-650M® and the chromatographic gel or resin was preequilibratedwith the same Tris buffer as used for resuspension before applying thefibrinogen solution. Loosely bound substances were washed out with theequilibration buffer followed by washing with a wash buffer (1.5 g/lsodium citrate, 6.0 g/l sodium chloride, adjusted to about pH 7.0 and aconductivity of about 12.0 mS/cm). Fibrinogen was then eluted from thechromatographic column with an elution buffer (1.5 g/l sodium citrate,and 10.0 g/l glycine adjusted to the same pH as the washing buffer andadjusted with about 7.0 g/l NaCl to the conductivity of 13.1-15 mS/cm).Nanofiltration was performed by successive passage of fibrinogensolution over nanofilters of decreasing pore size from 75 nm down to <35nm.

The resulting fibrinogen solution was concentrated, formulated andsterile filtered. This sterile filtered bulk was stored for 5 days at−80° C. before being sterile filtered for a second time and filled intofinal containers. One part of final containers was lyophilised while theother part was kept as a liquid formulation. No detectable amounts ofchelating agents were observed in the lyophilised product or the liquidconcentrate.

Reconstitution of lyophilisates was accomplished by addition of waterfor injection (WFI) up to the concentration before lyophilisation.

Examples II-XII were performed in the same way as example I butcomprised variation of type and concentration of chelating agents aswell as variations of extraction time. While parameters like proteincontent, fibrinogen-antigen content or fibrinopeptide-A content were notsignificantly influenced by these variations when being normalised to 1mg fibrinogen, it was observed that the content of compounds of highermolecular weight than fibrinogen (HMW), determined by size exclusionchromatography, exceeded 10% when the concentration of the complexingagent was less than 3 mmol/l. Example XIII was prepared according to theprocess of WO-A1-2012/038410, i.e. without any chelating agent presentduring purification. The outcome of these variations is summarised intable 1.

TABLE 1 Example Substance mmol/l HMW % II EDTA 1 18 III EDTA 3 10 IVEDTA 5 4 V EDTA 20 4 VI EDTA 50 4 XIII — 0 20 XI EGTA 1 13 XII BAPTA 122 IX EGTA 5 3 X BAPTA 5 6 I EDTA 10 3 VII EGTA 10 2 VIII BAPTA 10 4

A set of experiments was performed to determine a suitable extractiontime range as a compromise between yield and purity of the extractedfibrinogen intermediate. The suitable extraction time range wasdetermined to be between 10 to 120 minutes as less extraction timeprovided a very pure intermediate at the cost of fibrinogen yield, whileat extraction times exceeding 120 minutes it was observed that someimpurities began to redissolve without a significant gain in fibrinogenyield.

Comparison with WO-A1-2012/038410.

A difference between the present invention and WO-A1-2012/038410 isrepresented by the addition of a chelating agent prior to precipitationof fibrinogen by a suitable precipitation agent, like glycine, andreplacement of the following resuspension step in WO-A1-2012/038410 byan extraction. Said modification resulted in an unexpected increase ofcoagulation factor XIII activity in the final product of the presentinvention, i.e. up to 0.30 IU/mg fibrinogen (fibrinogen concentration20-25 mg/ml; determined by the Clauss method), as well as an increasedyield.

FIG. 1 depicts a SDS-Page at non-reducing conditions revealing less highmolecular weight compounds in typical products produced according to theprocess of the present invention (Lanes 6-11 also indicated as “+”)compared to the product of patent application WO-A1-2012/038410 (Lanes2-5 also indicated as “−”). The protein band closest to 250 kDrepresents fibrinogen while those above the fibrinogen band arecompounds of higher molecular weight. The protein band at about 50 kDrepresents albumin. Lane 1 displays the molecular weight markers.

FIG. 2 depicts a SDS-Page at reducing conditions. Tested products arethe same as in FIG. 1 and in the same order and they are consequentlyindicated in the same manner as in FIG. 1, i.e. “+” for productsprepared by a process according to the present invention, while “−”indicates products prepared by a process according to WO-A1-2012/038410.The major bands at about 50-70 kD represent the α-, β- and γ-chains offibrinogen. The protein band at about 100 kD represent the dimer of thefibrinogen γ-chain. The faint band at about 30 kD is caused byfibrinogen fragments. Lane 1 displays the molecular weight markers.

Comparison with WO-A2-2009/155626

Differences between the products of the present invention and those ofWO-A2-2009/155626 were investigated by analysis of products prepared bythe processes of WO-A2-2009/155626, in particular by combination ofdisclosed examples 1 and 6, which results in a nanofiltered andlyophilised product. It was observed that the product ofWO-A2-2009/155626 contained 1% of compounds of higher molecular weightthan fibrinogen, determined by size exclusion chromatography, and acoagulation factor XIII activity of about 0.41 IU/mg fibrinogen.

The invention claimed is:
 1. A pharmaceutical fibrinogen solutionproduct comprising 2% to 6% of compounds of higher molecular weight thanfibrinogen, with fibrinogen having an apparent molecular weight of 340kD, determined as % of total area by size exclusion chromatography at280 nm; a coagulation factor XIII activity in concentrations of0.05-0.30 IU/mg fibrinogen; and having no detectible amount ofantithrombin-III (AT-III) and no detectible amount of proteolyticactivity as measured by an AT-III concentration of less than 0.2 IU/mland a proteolytic activity of less than 2 U per litre (<2 U/l), whichequates to less than 0.01 IU AT-III per mg fibrinogen and less than 0.1mU proteolytic activity per mg fibrinogen as measured in a solution ofthe product containing fibrinogen in a concentration of 20 mg/mL;wherein said fibrinogen solution product is obtained by a processcomprising precipitation of fibrinogen by a precipitating agent from afibrinogen containing solution in the presence of one or more chelatingagent(s) to form a fibrinogen paste and removal of the supernatant fromthe fibrinogen paste, characterised in that fibrinogen is extracted fromthe paste forming a liquid fraction containing fibrinogen, and anundissolved residue, which is separated from the liquid, withoutaddition of one or more protease inhibitor(s).
 2. The pharmaceuticalfibrinogen solution product according to claim 1, wherein the processcomprises precipitating the fibrinogen with the precipitating agent fromthe fibrinogen containing source in the presence of the one or morechelating agent(s) to form the fibrinogen paste, removing thesupernatant from the fibrinogen paste, extracting the fibrinogen fromthe fibrinogen paste in an aqueous medium void of the chelating agent(s)for a suitable extraction time thereby forming the liquid fractioncontaining fibrinogen and the undissolved residue, and separating theundissolved residue from the liquid fraction containing fibrinogen,wherein addition of one or more protease inhibitor(s) is omitted in allsteps of the process, wherein the fibrinogen is precipitated in atemperature range of from 4.1° C. to 40° C., and the one or moreprotease inhibitor(s) is selected from the group consisting ofC1-protease inhibitors, trypsin inhibitors, thrombin inhibitors,antithrombin-III (AT-III), heparin-cofactor-II, aprotinin, pepstatin,leupeptin and epsilon-aminocaproic acid.
 3. The pharmaceuticalfibrinogen solution product according to claim 1, wherein theconcentration of the chelating agent in the water phase is 3 mM to 100mM.
 4. The pharmaceutical fibrinogen solution product according to claim1, wherein the process further comprises the steps of: a) solubilising acryoprecipitate, solubilised at about neutral pH to form the solution;b) subjecting the solution to adsorption with Al(OH)₃ and removing aresulting gel; c) virus inactivating the resulting solution of step b)by a solvent/detergent (S/D) treatment, extraction of S/D reagents withvegetable oil and contacting a water-phase with a TMAE resin at apH-value of 6.9-7.1 and an osmolality of 570-610 mosmol/l; d) adding atleast one chelating agent to the resulting water phase of step c),wherein a concentration of the chelating agent in the water phase is 3mM to 100 mM; e) precipitating the fibrinogen from the chelatingagent-containing water phase from step d) by adding glycine until afinal concentration of about 1M glycine is reached to form thefibrinogen paste, and separating the resulting fibrinogen paste; f)extracting the fibrinogen from the fibrinogen paste by a 20 mM TRISbuffer at a pH of about 8.0 and filtering to form a filtered solution;g) loading the filtered solution of step f) onto an anion exchange resincomprising trimethyl-amino groups grafted to a hydroxylated methacrylicpolymer backbone via linking groups and washing off loosely boundsubstances with a wash buffer of about 12.0 mS/cm conductivity; h)eluting the fibrinogen with an elution buffer containing about 1.5 g/lsodium citrate, about 7.0 g/l sodium chloride, and about 10.0 g/lglycine, the elution buffer adjusted to a pH of about 7.0 and aconductivity of 13.1-15 mS/cm; i) filtering the fibrinogen over at leastone nanofilter; and j) concentrating, formulating, sterile filtering,filling into suitable containers, and optionally lyophilising thefibrinogen; wherein addition of one or more protease inhibitor(s) isomitted, and the fibrinogen product having less than 2%-6% of compoundsof higher molecular weight than fibrinogen, determined as % of totalarea by size exclusion chromatography at 280 nm.
 5. A pharmaceuticalfibrinogen solution product obtained by a process comprisinglyophilizing the fibrinogen solution product of claim
 1. 6. Thepharmaceutical fibrinogen solution product according to claim 4, whereinthe anion exchange resin comprises a support material comprising ahydroxylated polymer backbone with grafted tertiary or quaternary aminogroups.
 7. The pharmaceutical fibrinogen solution product of claim 5,which, when reconstituted with water to a concentration of 20 mg/mL,comprises: 2% to 6% of compounds of higher molecular weight thanfibrinogen, determined as % of total area by size exclusionchromatography at 280 nm; a coagulation factor XIII activity inconcentrations of 0.05-0.30 IU/mg fibrinogen; and has no detectibleamount of antithrombin-III (AT-III) and no detectible amount ofproteolytic activity as measured by an AT-III concentration of less than0.2 IU/mL and a proteolytic activity of less than 2 U per litre (<2U/l), which equates to less than 0.01 IU AT-III per mg fibrinogen andless than 0.1 mU proteolytic activity per mg fibrinogen.